SCN5A (Nav1.5): Predicting the Consequence of Missense Single- Nucleotide Polymorphisms.

SCN5A (Nav1.5)：预测错义单核苷酸多态性的后果。

• 批准号: 9224146
• 负责人: Brett M Kroncke
• 金额: $ 12.25万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9224146
• 关键词: Action Potentials; Algorithms; Amino Acid Sequence; Amino Acids; Apical; Award; Benign; Biological Models; Biology; Brugada syndrome; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cell surface; Cells; Characteristics; Chemistry; Chlorides; Clinical; Collaborations; Computer Simulation; Data; Data Set; Defect; Dilated Cardiomyopathy; Discipline; Discrimination; Disease; Education; Electrophysiology (science); Environment; Equilibrium; Estrogens; Evaluation; Family; Fluorescence-Activated Cell Sorting; Foundations; Genes; Genetic Variation; Genomic medicine; Goals; Heart Diseases; Hip region structure; Human; Induced Mutation; Ion Channel; Laboratories; Learning; Link; Literature; Long QT Syndrome; Membrane Proteins; Mentors; Methods; Missense Mutation; Modeling; Mutation; Nature; Noise; Nuclear Magnetic Resonance; Output; Pathogenicity; Penetrance; Phase; Phenotype; Point Mutation; Postdoctoral Fellow; Production; Proteins; Recovery; Research; Research Personnel; Research Project Grants; Resources; Risk Factors; Schools; Scientist; Sick Sinus Syndrome; Signal Transduction; Single Nucleotide Polymorphism; Sodium; Sodium Channel; Spectrum Analysis; Structure; Syndrome; Techniques; Technology; Testing; Time; Training; Translational Research; Transmembrane Domain; Universities; Validation; Variant; Virginia; base; career development; clinical Diagnosis; density; flexibility; genetic variant; genomic profiles; human disease; improved; induced pluripotent stem cell; instrument; molecular dynamics; next generation sequencing; personalized medicine; prediction algorithm; predictive modeling; prevent; profiles in patients; protein function; protein structure; skills; structural biology; tool; trafficking; undergraduate student; variant of unknown significance; voltage

Project Summary/Abstract Candidate Background: In graduate school at the University of Virginia, I built on my undergraduate spectroscopy education by using spectroscopic tools to investigate membrane protein flexibility. As a Postdoctoral Fellow at Vanderbilt, I transitioned to membrane protein structural biology involved in human disease, specifically KCNQ and KCNE family-associated channelopathies. As a Postdoctoral Fellow, I have been involved in several projects concerning the structural underpinnings of disease mechanisms, most recently proposing a mechanism for diminished apical chloride secretion through an estrogen-induced loss of KCNQ1- KCNE3 channel conduction. Research Strategy: The human voltage-gated sodium channel Nav1.5 (encoded by SCN5A) is implicated in several diseases of the heart including dilated cardiomyopathy, cardiac conduction disease, sick sinus syndrome, type 3 longQT syndrome, and Brugada syndrome. Several algorithms accurately predict SCN5A variants that are ultimately harmful (SIFT, PolyPhen-2, PredSNP, etc.). However, there is a significant gap in the negative predictive ability of these methods, i.e. the ability to accurately classify a variant as benign. The approach I am proposing is to tackle this problem on two fronts: 1) incorporating channel-specific, quantitative information-rich data into predictive model construction—the objective being to predict channel function, instead of disease- inducing propensity—and 2) including a set of point mutation variants enriched in WT/neutral phenotypes to improve discrimination power during model training and evaluation. This project aims to ultimately predict Nav1.5 channel phenotypes for all possible amino-acid changing single nucleotide polymorphisms (nsSNP) by balancing high-throughput computation and rigorous experimental validation with model systems: predicting the nearly 15,000 possible SCN5A missense nsSNPs is currently only feasible in silico, i.e. leveraging calculable channel-specific protein sequence and structure-based features. The availability of a high-throughput electrophysiology instrument allows for an unprecedented amassing of ion channel functional output from heterologously expressed Nav1.5; the evaluation of SCN5A variants impact on action potential in the more native like human induced pluripotent stem cell cardiomyocytes is possible in low-throughput. During the mentored (K99) phase of this award, I will generate (mis)trafficking and electrophysiology current output data from missense nsSNPs of SCN5A, focusing on the Voltage-Sensing Module (VSM) of domain IV (Aim 1) and train an SCN5A VSM IV-specific phenotype prediction model using trafficking and electrophysiology data from Aim 1 and the literature (Aim 2). As an independent investigator, I will determine structure and flexibility-induced changes from selected variants using a combination of Rosetta modeling and nuclear magnetic resonance (NMR) to refine the predictive model (Aim 3). Career Development and Training: My training proposal is ambitious covering several disciplines, some of which will be new to me. The skills I will acquire are developing computational predictive models of ion channel phenotypes, trafficking/expression quantitation through Fluorescence Activated Cell Sorting (FACS), CRISPR/Cas9 gene manipulation, and hiPSC cardiomyocyte production. Though there are many activities planned, I will be trained directly in the laboratories of prominent scientists in their respective fields: Charles Sanders, Jens Meiler, and Dan Roden.

项目总结/文摘